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. 1994 Sep 13;91(19):8950–8954. doi: 10.1073/pnas.91.19.8950

Cyanotriphenylborate: subtype-specific blocker of glycine receptor chloride channels.

N Rundström 1, V Schmieden 1, H Betz 1, J Bormann 1, D Langosch 1
PMCID: PMC44724  PMID: 8090751

Abstract

The inhibitory glycine receptor is a ligand-gated ion-channel protein existing in different homo- and heterooligomeric isoforms. Here we show that the chloride channel of the recombinant alpha 1-subunit homooligomeric glycine receptor is efficiently blocked by cyanotriphenylborate (CTB) with a concentration effecting 50% inhibition (IC50) of 1.3 microM in the presence of 50 microM glycine. The antagonistic effect of CTB is noncompetitive, use dependent, and more pronounced at positive membrane potentials, suggesting open-channel block. In contrast to alpha 1-subunit receptors, alpha 2-subunit homooligomers are resistant to CTB (IC50 >> 20 microM). By exchanging the channel-lining transmembrane segment M2 of the alpha 1 polypeptide by that of the alpha 2 polypeptide, we could transfer this resistance to alpha 1 channels, indicating that a single glycine residue at position 254 of the alpha 1 subunit is critical for CTB sensitivity. The blocker did not affect the cation-selective channel of the nicotinic acetylcholine receptor. Thus, CTB may prove useful as a tool to probe the subunit structure of native glycine receptors in mammalian neurons.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Becker C. M., Hoch W., Betz H. Glycine receptor heterogeneity in rat spinal cord during postnatal development. EMBO J. 1988 Dec 1;7(12):3717–3726. doi: 10.1002/j.1460-2075.1988.tb03255.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bormann J., Hamill O. P., Sakmann B. Mechanism of anion permeation through channels gated by glycine and gamma-aminobutyric acid in mouse cultured spinal neurones. J Physiol. 1987 Apr;385:243–286. doi: 10.1113/jphysiol.1987.sp016493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bormann J., Rundström N., Betz H., Langosch D. Residues within transmembrane segment M2 determine chloride conductance of glycine receptor homo- and hetero-oligomers. EMBO J. 1993 Oct;12(10):3729–3737. doi: 10.1002/j.1460-2075.1993.tb06050.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Charnet P., Labarca C., Leonard R. J., Vogelaar N. J., Czyzyk L., Gouin A., Davidson N., Lester H. A. An open-channel blocker interacts with adjacent turns of alpha-helices in the nicotinic acetylcholine receptor. Neuron. 1990 Jan;4(1):87–95. doi: 10.1016/0896-6273(90)90445-l. [DOI] [PubMed] [Google Scholar]
  5. Chen C., Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. doi: 10.1128/mcb.7.8.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cohen B. N., Labarca C., Davidson N., Lester H. A. Mutations in M2 alter the selectivity of the mouse nicotinic acetylcholine receptor for organic and alkali metal cations. J Gen Physiol. 1992 Sep;100(3):373–400. doi: 10.1085/jgp.100.3.373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ffrench-Constant R. H., Rocheleau T. A., Steichen J. C., Chalmers A. E. A point mutation in a Drosophila GABA receptor confers insecticide resistance. Nature. 1993 Jun 3;363(6428):449–451. doi: 10.1038/363449a0. [DOI] [PubMed] [Google Scholar]
  8. Grenningloh G., Pribilla I., Prior P., Multhaup G., Beyreuther K., Taleb O., Betz H. Cloning and expression of the 58 kd beta subunit of the inhibitory glycine receptor. Neuron. 1990 Jun;4(6):963–970. doi: 10.1016/0896-6273(90)90149-a. [DOI] [PubMed] [Google Scholar]
  9. Grenningloh G., Schmieden V., Schofield P. R., Seeburg P. H., Siddique T., Mohandas T. K., Becker C. M., Betz H. Alpha subunit variants of the human glycine receptor: primary structures, functional expression and chromosomal localization of the corresponding genes. EMBO J. 1990 Mar;9(3):771–776. doi: 10.1002/j.1460-2075.1990.tb08172.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hamill O. P., Marty A., Neher E., Sakmann B., Sigworth F. J. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 1981 Aug;391(2):85–100. doi: 10.1007/BF00656997. [DOI] [PubMed] [Google Scholar]
  11. Hucho F., Oberthür W., Lottspeich F. The ion channel of the nicotinic acetylcholine receptor is formed by the homologous helices M II of the receptor subunits. FEBS Lett. 1986 Sep 1;205(1):137–142. doi: 10.1016/0014-5793(86)80881-x. [DOI] [PubMed] [Google Scholar]
  12. Imoto K., Busch C., Sakmann B., Mishina M., Konno T., Nakai J., Bujo H., Mori Y., Fukuda K., Numa S. Rings of negatively charged amino acids determine the acetylcholine receptor channel conductance. Nature. 1988 Oct 13;335(6191):645–648. doi: 10.1038/335645a0. [DOI] [PubMed] [Google Scholar]
  13. Imoto K., Konno T., Nakai J., Wang F., Mishina M., Numa S. A ring of uncharged polar amino acids as a component of channel constriction in the nicotinic acetylcholine receptor. FEBS Lett. 1991 Sep 9;289(2):193–200. doi: 10.1016/0014-5793(91)81068-j. [DOI] [PubMed] [Google Scholar]
  14. Langosch D., Becker C. M., Betz H. The inhibitory glycine receptor: a ligand-gated chloride channel of the central nervous system. Eur J Biochem. 1990 Nov 26;194(1):1–8. doi: 10.1111/j.1432-1033.1990.tb19419.x. [DOI] [PubMed] [Google Scholar]
  15. Lauffer L., Hucho F. Triphenylmethylphosphonium is an ion channel ligand of the nicotinic acetylcholine receptor. Proc Natl Acad Sci U S A. 1982 Apr;79(7):2406–2409. doi: 10.1073/pnas.79.7.2406. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Malosio M. L., Marquèze-Pouey B., Kuhse J., Betz H. Widespread expression of glycine receptor subunit mRNAs in the adult and developing rat brain. EMBO J. 1991 Sep;10(9):2401–2409. doi: 10.1002/j.1460-2075.1991.tb07779.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Pribilla I., Takagi T., Langosch D., Bormann J., Betz H. The atypical M2 segment of the beta subunit confers picrotoxinin resistance to inhibitory glycine receptor channels. EMBO J. 1992 Dec;11(12):4305–4311. doi: 10.1002/j.1460-2075.1992.tb05529.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Revah F., Galzi J. L., Giraudat J., Haumont P. Y., Lederer F., Changeux J. P. The noncompetitive blocker [3H]chlorpromazine labels three amino acids of the acetylcholine receptor gamma subunit: implications for the alpha-helical organization of regions MII and for the structure of the ion channel. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4675–4679. doi: 10.1073/pnas.87.12.4675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Schmieden V., Grenningloh G., Schofield P. R., Betz H. Functional expression in Xenopus oocytes of the strychnine binding 48 kd subunit of the glycine receptor. EMBO J. 1989 Mar;8(3):695–700. doi: 10.1002/j.1460-2075.1989.tb03428.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Spivak C. E., Albuquerque E. X. Triphenylmethylphosphonium blocks the nicotinic acetylcholine receptor noncompetitively. Mol Pharmacol. 1985 Feb;27(2):246–255. [PubMed] [Google Scholar]
  21. Takahashi T., Momiyama A., Hirai K., Hishinuma F., Akagi H. Functional correlation of fetal and adult forms of glycine receptors with developmental changes in inhibitory synaptic receptor channels. Neuron. 1992 Dec;9(6):1155–1161. doi: 10.1016/0896-6273(92)90073-m. [DOI] [PubMed] [Google Scholar]
  22. Unwin N. Neurotransmitter action: opening of ligand-gated ion channels. Cell. 1993 Jan;72 (Suppl):31–41. doi: 10.1016/s0092-8674(05)80026-1. [DOI] [PubMed] [Google Scholar]
  23. Villarroel A., Herlitze S., Koenen M., Sakmann B. Location of a threonine residue in the alpha-subunit M2 transmembrane segment that determines the ion flow through the acetylcholine receptor channel. Proc Biol Sci. 1991 Jan 22;243(1306):69–74. doi: 10.1098/rspb.1991.0012. [DOI] [PubMed] [Google Scholar]
  24. Witzemann V., Stein E., Barg B., Konno T., Koenen M., Kues W., Criado M., Hofmann M., Sakmann B. Primary structure and functional expression of the alpha-, beta-, gamma-, delta- and epsilon-subunits of the acetylcholine receptor from rat muscle. Eur J Biochem. 1990 Dec 12;194(2):437–448. doi: 10.1111/j.1432-1033.1990.tb15637.x. [DOI] [PubMed] [Google Scholar]

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